Methods of modulating mitochondrial nad-dependent deacetylase

ABSTRACT

The present invention provides methods for identifying agents that modulate a level or an activity of a mitochondrial NAD-dependent deacetylase polypeptide, as well as agents identified by the methods. The invention further provides methods of modulating mitochondrial NAD-dependent deacetylase activity in a cell. The invention further provides methods of modulating mitochondrial function by modulating the activity of mitochondrial NAD-dependent deacetylase.

FIELD OF THE INVENTION

The present invention is in the field of deacetylase enzymes.

BACKGROUND OF THE INVENTION

Silent Information Regulator 2 (Sir2) protein is involved intranscriptional silencing and DNA damage repair in yeast. It alsoincreases life span in yeast and in Caenorhabditis elegans. Yeast Sir2protein has an NAD-dependent histone deacetylase activity that linksSir2 functions to cellular metabolism. This NAD-dependent deacetylaseactivity is conserved from bacteria to humans and mammalian Sir2homologues also have NAD-dependent histone deacetylase activity. TheNAD-dependency of Sir2-like enzymes distinguishes them from the class Iand II HDAC histone deacetylases that use a zinc-catalyzed mechanism.Seven Sir2 homologues have been identified in humans and are designatedhSIRT1-7. Among the human homologues, hSIRT1, hSIRT2 and hSIRT3 the mosthomologous to yeast Sir2 and have NAD-dependent deacetylase activity. Atpresent, very little is known about the in vivo functions of mammalianSIR2 homologues. hSIRT1 deacetylates the transcription factor p53thereby inhibiting p53 activation and apoptosis in response to DNAdamage and oxidative stress.

Literature

Yang et al. (2000) Genomics 69:355-369; Frye (2000)Biochem. Biophys.Res. Comm. 273:793-798; GenBank Accession Nos. NM_(—)012239 andAF083109.

SUMMARY OF THE INVENTION

The present invention provides methods for identifying agents thatmodulate a level or an activity of a mitochondrial NAD-dependentdeacetylase polypeptide, as well as agents identified by the methods.The invention further provides methods of modulating mitochondrialNAD-dependent deacetylase activity in a cell. The invention furtherprovides methods of modulating mitochondrial function by modulating theactivity of mitochondrial NAD-dependent deacetylase.

FEATURES OF THE INVENTION

The present invention features an in vitro method of identifying anagent that modulates an enzymatic activity of a human mitochondrialNAD-dependent deacetylase. The method generally involves contacting amitochondrial NAD-dependent deacetylase polypeptide with a test agent inan assay mixture that comprises nicotinamide adenine dinucleotide (NAD)and an acetylated histone peptide; and determining the effect, if any,of the test agent on the enzymatic activity of mitochondrialNAD-dependent deacetylase. In some embodiments, the human mitochondrialNAD-dependent deacetylase polypeptide comprises an amino acid sequenceas set forth in SEQ ID NO:01. In some embodiments, the acetylatedhistone peptide comprises amino acids 1-22 of histone 4. In someembodiments, the acetylated histone peptide contains a ¹⁴C-labeledacetyl group, and determining the effect of the agent on the enzymaticactivity of the deacetylase is performed by measuring release of theradioactive acetyl group. In other embodiments, determining the effectof the agent on the enzymatic activity of the deacetylase is performedby detecting binding of an antibody specific for acetylated histone.

The present invention further features an in vitro method foridentifying an agent that modulates a level of mitochondrialNAD-dependent deacetylase in a cell. The method generally involvescontacting a cell that produces mitochondrial NAD-dependent deacetylasewith a test agent; and determining the effect, if any, of the test agenton the level of mitochondrial NAD-dependent deacetylase. In someembodiments, determining the effect of the agent on the level of thedeacetylase is performed by determining a level of mitochondrialNAD-dependent deacetylase mRNA in the cell. In other embodiments,determining the effect of the agent on the level of the deacetylase isperformed by determining a level of mitochondrial NAD-dependentdeacetylase polypeptide in the cell.

The present invention further features a biologically active agentidentified by a screening method according to the invention. Theinvention further features a pharmaceutical composition comprising abiologically active agent that reduces a level or an activity of amitochondrial NAD-dependent deacetylase protein; and a pharmaceuticallyacceptable excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-F depict deacetylase activity in mitochondria.

FIGS. 2A-C depict localization of deacetylase activity in mitochondria.

FIG. 3 depicts depict requirement of the N-terminal region of hSIRT3 formitochondrial targeting.

FIGS. 4A-C depict mitochondrial import of hSIRT3.

FIGS. 5A and 5B depict intramitochondrial localization of hSIRT3.

FIGS. 6A-C depict proteolytic processing of hSIRT3 by MPP.

FIG. 7 depicts the amino acid sequence of human SIRT3 (SEQ ID NO:01).

FIG. 8 depicts NAD-dependent HDAC activity of truncated recombinantSIRT3.

DEFINITIONS

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymeric form of amino acids of any length, which caninclude coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and homologous leader sequences, with orwithout N-terminal methionine residues; immunologically tagged proteins;and the like.

A “substantially isolated” or “isolated” polypeptide is one that issubstantially free of the macromolecules with which it is associated innature. By substantially free is meant at least 50%, preferably at least70%, more preferably at least 80%, and even more preferably at least 90%free of the materials with which it is associated in nature.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such aspolynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

The term “disorder associated with mitochondrial malfunction,” as usedherein, refers to any disorder that is directly or indirectly a resultof, or caused by, malfunction of a mitochondrion in a cell.

The terms “cancer”, “neoplasm”, “tumor”, and “carcinoma”, are usedinterchangeably herein to refer to cells which exhibit relativelyautonomous growth, so that they exhibit an aberrant growth phenotypecharacterized by a significant loss of control of cell proliferation.Cancerous cells can be benign or malignant.

As used herein, the terms “treatment”, “treating”, and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, i.e., causing regression of the disease.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “amitochondrial NAD-dependent deacetylase” includes a plurality of suchdeacetylases and reference to “the agent” includes reference to one ormore agents and equivalents thereof known to those skilled in the art,and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention provides methods for identifying agents thatmodulate an enzymatic activity of a mitochondrial NAD-dependentdeacetylase. The invention further provides agents identified by theinstant methods, as well as methods of modulating the activity of themitochondrial NAD-dependent deacetylase. Agents that modulate theactivity of an NAD-dependent mitochondrial deacetylase are useful toameliorate disorders associated with mitochondrial malfunction.

The instant invention is based on the observation that human SIRT3(hSIRT3) is a nuclear-encoded NAD-dependent deacetylase residing withinthe mitochondria. hSIRT3 is proteolytically cleaved by a mitochondrialenzyme to a catalytically active form of having a molecular weight ofabout 28 kDa. The identification of the activity of hSIRT3 alloweddevelopment of assays to identify agents that modulate the activity ofthis enzyme. Such agents are useful in treating disorders arising frommitochondrial malfunction.

Screening Methods

The invention provides in vitro methods of identifying an agent thatmodulates a level or an activity of a mitochondrial NAD-dependentdeacetylase. The methods generally involve contacting a mitochondrialNAD-dependent deacetylase protein, or a cell that produces amitochondrial NAD-dependent deacetylase protein, with a test agent, anddetermining the effect, if any, on a level or an activity of themitochondrial NAD-dependent deacetylase protein.

In some embodiments, the methods are cell-free methods. Cell-freemethods generally involve contacting a mitochondrial NAD-dependentdeacetylase with a test agent and determining the effect, if any, of thetest agent on the enzymatic activity of the mitochondrial NAD-dependentdeacetylase.

In other embodiments, the methods are cell-based methods. Cell-basedmethods generally involve contacting a cell that produces mitochondrialNAD-dependent deacetylase with a test agent and determining the effect,if any, of the test agent on the level of mitochondrial NAD-dependentdeacetylase mRNA or mitochondrial NAD-dependent deacetylase protein inthe cell.

As used herein, the term “determining” refers to both quantitative andqualitative determinations and as such, the term “determining” is usedinterchangeably herein with “assaying,” “measuring,” and the like.

The term “mitochondrial NAD-dependent deacetylase polypeptide”encompasses human mitochondrial NAD-dependent deacetylase proteins(e.g., human SIRT3 proteins) having the amino acid sequences set forthin any of GenBank Accession Nos. NM_(—)012239; and AF0831087, where thepolypeptide is a nuclear-encoded, mitochondrial protein and exhibitsNAD-dependent mitochondrial NAD-dependent deacetylase activity. The termcomprises a mitochondrial NAD-dependent deacetylase polypeptidecomprises the amino acid sequence as set forth in SEQ ID NO:01 anddepicted in FIG. 7; catalytically active fragments thereof; andcatalytically active variants thereof. Catalytically active fragmentsinclude fragments lacking from about 1 to about 120 N-terminal aminoacids of the sequence set forth in SEQ ID NO:01. For example,catalytically active fragments lacking from about 1 to about 10, fromabout 10 to about 20, from about 20 to about 30, from about 30 to about40, from about 40 to about 50, from about 50 to about 60, from about 60to about 70, from about 70 to about 80, from about 80 to about 90, fromabout 90 to about 100, or from about 100 to about 120 N-terminal aminoacids of the sequence set forth in SEQ ID NO:01 can be used in a subjectmethod. The term encompasses an enzyme that is proteolytically processedin the mitochondria by a mitochondrial enzyme referred to as MPP, whichcleaves the 44 kDa form of the enzyme to a catalytically active 28 kDaform. In many embodiments, the 28 kDa form is used in the instantmethods. The term encompasses variants that have insertions, deletions,and/or conservative amino acid substitutions that do not affect theability of the protein to deacetylate an appropriate substrate (e.g.,acetylated histone 4, or an acetylated fragment thereof) in anNAD-dependent fashion. In some embodiments, the mitochondrialNAD-dependent deacetylase is recombinant, e.g., produced in a celltransfected with an expression construct comprising a nucleotidesequence that encodes the mitochondrial NAD-dependent deacetylase.

The term “mitochondrial NAD-dependent deacetylase polypeptide” furtherencompasses fusion proteins comprising a mitochondrial NAD-dependentdeacetylase and a heterologous polypeptide (“fusion partners”), wheresuitable fusion partners include immunological tags such as epitopetags, including, but not limited to, hemagglutinin, FLAG, and the like;proteins that provide for a detectable signal, including, but notlimited to, fluorescent proteins (e.g., a green fluorescent protein, afluorescent protein from an Anthozoan species, and the like), enzymes(e.g., β-galactosidase, luciferase, horse radish peroxidase, etc.), andthe like; polypeptides that facilitate purification or isolation of thefusion protein, e.g., metal ion binding polypeptides such as 6His tags(e.g., mitochondrial NAD-dependent deacetylase/6His), GST, and the like.The term “mitochondrial NAD-dependent deacetylase polypeptide” furtherincludes a mitochondrial NAD-dependent deacetylase polypeptide modifiedto include one or more specific protease cleavage sites.

Where the assay is an in vitro cell-free assay, the methods generallyinvolve contacting a mitochondrial NAD-dependent deacetylase polypeptidewith a test agent. The mitochondrial NAD-dependent deacetylasepolypeptide may be, but need not be, purified. For example, themitochondrial NAD-dependent deacetylase polypeptide can be in a celllysate, or may be isolated, or partially purified. Thus, the assay canbe conducted in the presence of additional components, as long as theadditional components do not adversely affect the reaction to anunacceptable degree.

Where the assay is an in vitro cell-based assay, any of a variety ofcells can be used. The cells used in the assay are usually eukaryoticcells, including, but not limited to, rodent cells, human cells, andyeast cells. The cells may be primary cell cultures or may beimmortalized cell lines. The cells may be “recombinant,” e.g., the cellmay have transiently or stably introduced therein a construct (e.g., aplasmid, a recombinant viral vector, or any other suitable vector) thatcomprises a nucleotide sequence encoding a mitochondrial NAD-dependentdeacetylase polypeptide, or that comprises a nucleotide sequence thatcomprises a mitochondrial NAD-dependent deacetylase promoter operablylinked to a reporter gene.

The terms “candidate agent,” “test agent,” “agent”, “substance” and“compound” are used interchangeably herein. Candidate agents encompassnumerous chemical classes, typically synthetic, semi-synthetic, ornaturally occurring inorganic or organic molecules. Candidate agentsinclude those found in large libraries of synthetic or naturalcompounds. For example, synthetic compound libraries are commerciallyavailable from Maybridge Chemical Co. (Trevillet, Cornwall, UK),ComGenex (South San Francisco, Calif.), and MicroSource (New Milford,Conn.). A rare chemical library is available from Aldrich (Milwaukee,Wis.) and can also be used. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available from Pan Labs (Bothell, Wash.) or are readily producible.

Candidate agents may be small organic or inorganic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents may comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and mayinclude at least an amine, carbonyl, hydroxyl or carboxyl group, and maycontain at least two of the functional chemical groups. The candidateagents may comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Assays of the invention include controls, where suitable controlsinclude a sample (e.g., a sample comprising mitochondrial NAD-dependentdeacetylase protein, or a cell that synthesizes mitochondrialNAD-dependent deacetylase) in the absence of the test agent. Generally aplurality of assay mixtures is run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e. at zero concentration or below the level ofdetection.

Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Thecomponents of the assay mixture are added in any order that provides forthe requisite binding or other activity. Incubations are performed atany suitable temperature, typically between 4° C. and 40° C. Incubationperiods are selected for optimum activity, but may also be optimized tofacilitate rapid high-throughput screening. Typically between 0.1 and 1hour will be sufficient.

The screening methods may be designed a number of different ways, wherea variety of assay configurations and protocols may be employed, as areknown in the art. For example, one of the components may be bound to asolid support, and the remaining components contacted with the supportbound component. The above components of the method may be combined atsubstantially the same time or at different times.

Where the assay is a binding assay, following the contact and incubationsteps, the subject methods will generally, though not necessarily,further include a washing step to remove unbound components, where sucha washing step is generally employed when required to remove label thatwould give rise to a background signal during detection, such asradioactive or fluorescently labeled non-specifically bound components.Following the optional washing step, the presence of bound complexeswill then be detected.

A test agent of interest is one that reduces a level of mitochondrialNAD-dependent deacetylase protein or inhibits a mitochondrialNAD-dependent deacetylase activity by at least about 10%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 80%, at least about 90%, or more, when compared to a controlin the absence of the test agent.

Methods of Detecting Agents that Modulate a Level of MitochondrialNAD-Dependent Deacetylase mRNA and/or Mitochondrial NAD-DependentDeacetylase Polypeptide

The subject screening methods include methods of detecting an agent thatmodulates a level of a mitochondrial NAD-dependent deacetylase mRNAand/or mitochondrial NAD-dependent deacetylase polypeptide in a cell. Insome embodiments, the methods involve contacting a cell that producesmitochondrial NAD-dependent deacetylase with a test agent, anddetermining the effect, if any, of the test agent on the level ofmitochondrial NAD-dependent deacetylase mRNA in the cell.

A candidate agent is assessed for any cytotoxic activity it may exhibittoward the cell used in the assay, using well-known assays, such astrypan blue dye exclusion, an MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)assay, and the like. Agents that do not exhibit cytotoxic activity areconsidered candidate agents.

A wide variety of cell-based assays may be used for identifying agentswhich reduce a level of mitochondrial NAD-dependent deacetylase mRNA ina eukaryotic cell, using, for example, a cell that normally producesmitochondrial NAD-dependent deacetylase mRNA, a mammalian celltransformed with a construct comprising a mitochondrial NAD-dependentdeacetylase-encoding cDNA such that the cDNA is overexpressed, or,alternatively, a construct comprising a mitochondrial NAD-dependentdeacetylase promoter operably linked to a reporter gene.

Accordingly, the present invention provides a method for identifying anagent, particularly a biologically active agent, that reduces a level ofmitochondrial NAD-dependent deacetylase expression in a cell, the methodcomprising: combining a candidate agent to be tested with a cellcomprising a nucleic acid which encodes a mitochondrial NAD-dependentdeacetylase polypeptide, or a construct comprising a mitochondrialNAD-dependent deacetylase promoter operably linked to a reporter gene;and determining the effect of said agent on mitochondrial NAD-dependentdeacetylase expression. A decrease of at least about 10%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 80%, at least about 90%, or more, in the level (i.e., anamount) of mitochondrial NAD-dependent deacetylase mRNA and/orpolypeptide following contacting the cell with a candidate agent beingtested, compared to a control to which no agent is added, is anindication that the agent modulates mitochondrial NAD-dependentdeacetylase expression.

Mitochondrial NAD-dependent deacetylase mRNA and/or polypeptide whoselevels are being measured can be encoded by an endogenous mitochondrialNAD-dependent deacetylase polynucleotide, or the mitochondrialNAD-dependent deacetylase polynucleotide can be one that is comprisedwithin a recombinant vector and introduced into the cell, i.e., themitochondrial NAD-dependent deacetylase mRNA and/or polypeptide can beencoded by an exogenous mitochondrial NAD-dependent deacetylasepolynucleotide. For example, a recombinant vector may comprise anisolated mitochondrial NAD-dependent deacetylase transcriptionalregulatory sequence, such as a promoter sequence, operably linked to areporter gene (e.g., β-galactosidase, chloramphenicol acetyltransferase, a fluorescent protein, luciferase, or other gene that canbe easily assayed for expression).

In these embodiments, the method for identifying an agent that modulatesa level of mitochondrial NAD-dependent deacetylase expression in a cell,comprises: combining a candidate agent to be tested with a cellcomprising a nucleic acid which comprises a mitochondrial NAD-dependentdeacetylase gene transcriptional regulatory element operably linked to areporter gene; and determining the effect of said agent on reporter geneexpression. A recombinant vector may comprise an isolated mitochondrialNAD-dependent deacetylase transcriptional regulatory sequence, such as apromoter sequence, operably linked to sequences coding for amitochondrial NAD-dependent deacetylase polypeptide; or thetranscriptional control sequences can be operably linked to codingsequences for a mitochondrial NAD-dependent deacetylase fusion proteincomprising mitochondrial NAD-dependent deacetylase polypeptide fused toa polypeptide which facilitates detection. In these embodiments, themethod comprises combining a candidate agent to be tested with a cellcomprising a nucleic acid which comprises a mitochondrial NAD-dependentdeacetylase gene transcriptional regulatory element operably linked to amitochondrial NAD-dependent deacetylase polypeptide-coding sequence; anddetermining the effect of said agent on mitochondrial NAD-dependentdeacetylase expression, which determination can be carried out bymeasuring an amount of mitochondrial NAD-dependent deacetylase mRNA,mitochondrial NAD-dependent deacetylase polypeptide, or mitochondrialNAD-dependent deacetylase fusion polypeptide produced by the cell.

Cell-based assays generally comprise the steps of contacting the cellwith an agent to be tested, forming a test sample, and, after a suitabletime, assessing the effect of the agent on mitochondrial NAD-dependentdeacetylase expression. A control sample comprises the same cell withoutthe candidate agent added. Mitochondrial NAD-dependent deacetylaseexpression levels are measured in both the test sample and the controlsample. A comparison is made between mitochondrial NAD-dependentdeacetylase expression level in the test sample and the control sample.Mitochondrial NAD-dependent deacetylase expression can be assessed usingconventional assays. For example, when a mammalian cell line istransformed with a construct that results in expression of mitochondrialNAD-dependent deacetylase, mitochondrial NAD-dependent deacetylase mRNAlevels can be detected and measured, or mitochondrial NAD-dependentdeacetylase polypeptide levels can be detected and measured. A suitableperiod of time for contacting the agent with the cell can be determinedempirically, and is generally a time sufficient to allow entry of theagent into the cell and to allow the agent to have a measurable effecton mitochondrial NAD-dependent deacetylase mRNA and/or polypeptidelevels. Generally, a suitable time is between 10 minutes and 24 hours,or from about 1 hour to about 8 hours.

Methods of measuring mitochondrial NAD-dependent deacetylase mRNA levelsare known in the art, several of which have been described above, andany of these methods can be used in the methods of the present inventionto identify an agent which modulates mitochondrial NAD-dependentdeacetylase mRNA level in a cell, including, but not limited to, a PCR,such as a PCR employing detectably labeled oligonucleotide primers, andany of a variety of hybridization assays.

Similarly, mitochondrial NAD-dependent deacetylase polypeptide levelscan be measured using any standard method, several of which have beendescribed herein, including, but not limited to, an immunoassay such asenzyme-linked immunosorbent assay (ELISA), for example an ELISAemploying a detectably labeled antibody specific for a mitochondrialNAD-dependent deacetylase polypeptide.

Mitochondrial NAD-dependent deacetylase polypeptide levels can also bemeasured in cells harboring a recombinant construct comprising anucleotide sequence that encodes a mitochondrial NAD-dependentdeacetylase fusion protein, where the fusion partner provides for adetectable signal or can otherwise be detected. For example, where thefusion partner provides an immunologically recognizable epitope (an“epitope tag”), an antibody specific for an epitope of the fusionpartner can be used to detect and quantitate the level of mitochondrialNAD-dependent deacetylase. In some embodiments, the fusion partnerprovides for a detectable signal, and in these embodiments, thedetection method is chosen based on the type of signal generated by thefusion partner. For example, where the fusion partner is a fluorescentprotein, fluorescence is measured.

Fluorescent proteins suitable for use include, but are not limited to, agreen fluorescent protein (GFP), including, but not limited to, a“humanized” version of a GFP, e.g., wherein codons of thenaturally-occurring nucleotide sequence are changed to more closelymatch human codon bias; a GFP derived from Aequoria victoria or aderivative thereof, e.g., a “humanized” derivative such as Enhanced GFP,which are available commercially, e.g., from Clontech, Inc.; a GFP fromanother species such as Renilla reniformis, Renilla mulleri, orPtilosarcus guernyi, as described in, e.g., WO 99/49019 and Peelle etal. (2001) J. Protein Chem. 20:507-519; “humanized” recombinant GFP(hrGFP) (Stratagene); any of a variety of fluorescent and coloredproteins from Anthozoan species, as described in, e.g., Matz et al.(1999) Nature Biotechnol. 17:969-973; and the like. Where the fusionpartner is an enzyme that yields a detectable product, the product canbe detected using an appropriate means, e.g., β-galactosidase can,depending on the substrate, yield colored product, which is detectedspectrophotometrically, or a fluorescent product; luciferase can yield aluminescent product detectable with a luminometer; etc.

Agents that reduce a level of mitochondrial NAD-dependent deacetylaseprotein include agents that reduce a level of enzymatically activemitochondrial NAD-dependent deacetylase. In some embodiments, an agentthat reduces a level of enzymatically active mitochondrial NAD-dependentdeacetylase is an agent that inhibits activity of a mitochondrialprocessing peptidase (MPP). Whether MPP activity is inhibited can bedetermined using any known assay, e.g., detecting formation of the 28 kDactive form of mitochondrial NAD-dependent deacetylase.

A number of methods are available for analyzing nucleic acids for thepresence and/or level of a specific mRNA in a cell. The mRNA may beassayed directly or reverse transcribed into cDNA for analysis. Thenucleic acid may be amplified by conventional techniques, such as thepolymerase chain reaction (PCR), to provide sufficient amounts foranalysis. The use of the polymerase chain reaction is described inSaiki, et al. (1985), Science 239:487, and a review of techniques may befound in Sambrook, et al. Molecular Cloning: A Laboratory Manual, CSHPress 1989, pp. 14.2-14.33. Alternatively, various methods are known inthe art that utilize oligonucleotide ligation as a means of detectingpolymorphisms, for examples see Riley et al. (1990), Nucl. Acids Res.18:2887-2890; and Delahunty et al. (1996), Am. J. Hum. Genet.58:1239-1246.

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

A variety of different methods for determining the nucleic acidabundance in a sample are known to those of skill in the art, whereparticular methods of interest include those described in: Pietu et al.,Genome Res. (June 1996) 6: 492-503; Zhao et al., Gene (Apr. 24, 1995)156: 207-213; Soares, Curr. Opin. Biotechnol. (October 1997) 8: 542-546;Raval, J. Pharmacol Toxicol Methods (November 1994) 32: 125-127;Chalifour et al., Anal. Biochem (Feb. 1, 1994) 216: 299-304; Stolz &Tuan, Mol. Biotechnol. (December 19960 6: 225-230; Hong et al.,Bioscience Reports (1982) 2: 907; and McGraw, Anal. Biochem. (1984) 143:298. Also of interest are the methods disclosed in WO 97/27317, thedisclosure of which is herein incorporated by reference.

A number of methods are available for determining the expression levelof a gene or protein in a particular sample. For example, detection mayutilize staining of cells or histological sections with labeledantibodies, performed in accordance with conventional methods. Cells arepermeabilized to stain cytoplasmic molecules. The antibodies of interestare added to the cell sample, and incubated for a period of timesufficient to allow binding to the epitope, usually at least about 10minutes. The antibody may be labeled with radioisotopes, enzymes,fluorescers, chemiluminescers, or other labels for direct detection.Alternatively, a second stage antibody or reagent is used to amplify thesignal. Such reagents are well known in the art. For example, theprimary antibody may be conjugated to biotin, with horseradishperoxidase-conjugated avidin added as a second stage reagent. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. Alternatively, the secondary antibody conjugated to afluorescent compound, e.g. fluorescein, rhodamine, Texas red, etc. Theabsence or presence of antibody binding may be determined by variousmethods, including flow cytometry of dissociated cells, microscopy,radiography, scintillation counting, etc.

Methods of Detecting Agents that Modulate an Activity of a MitochondrialNAD-Dependent Deacetylase Polypeptide

Methods of detecting an agent that modulates an activity of amitochondrial NAD-dependent deacetylase polypeptide include cell-freeand cell-based methods. The methods generally involve contacting amitochondrial NAD-dependent deacetylase polypeptide with a test agentand determining the effect, if any, on the mitochondrial NAD-dependentdeacetylase enzyme activity.

The deacetylase activity of a mitochondrial NAD-dependent deacetylasecan be determined by incubating the enzyme in the presence of NAD and anacetylated substrate. Suitable acetylated substrates include acetylatedhistone 4, or a fragment thereof, e.g., amino acids 1-22 of histone 4.The amino acid sequence of amino acids 1-22 of histone 4 is:NH₂-MSGRGKGGKGLGKGGAKRHRKV-COOH (SEQ ID NO:02). Additional exemplarysuitable substrates include the following:NH₂-MSGRGKGGKGLGKGGAKRHRKVLRDNIQGI-COOH (from histone-4; SEQ ID NO:03);and NH₂-MARTKQTARKSTGGKAPRKQLATKAARKSA-COOH (from histone-3; SEQ IDNO:04). In the foregoing peptides, the acetylated lysine residues are initalics.

The acetylated histone peptide is present in the assay mixture at aconcentration of from about 20 μM to about 1 mM, from about 30 μM toabout 900 μM, from about 40 μM to about 700 μM, from about 50 μM toabout 500 μM, from about 50 μM to about 300 μM, or from about 60 μM toabout 100 μM. NAD is present in the assay mixture at a concentration ofabout 1 mM. The acetyl group on the histone peptide is radiolabeled,e.g., [¹⁴C]-acetyl is used. The assay then involves determining theamount of [¹⁴C]-acetyl that is released, typically by scintillationcounting. Other components, such as salts, reducing agents, and buffers,may be included.

In one exemplary embodiment, the enzymatic reaction mixture comprises 4mM MgCl₂, 0.2 mM DTT, 50 mM Tris-HCl, pH 9.0, amino acids 1-22 ofhistone 4, which peptide is aceylated with a radiolabel acetyl group,and 1 mM NAD.

Another method of detecting mitochondrial NAD-dependent deacetylaseactivity is to monitor the acetylation status of a histone substrateusing an antibody specific for acetylated histone substrate. Lack ofreactivity of the anti-acetylated histone antibody with the histonesubstrate indicates that the histone has been deacetylated. Thus, insome embodiments, the methods involve determining binding of ananti-acetylated histone antibody with the histone substrate.Anti-acetylated antibody/histone binding can be determined using anytype of immunological assay, including immunoblotting assays, ELISAassays, and the like.

In some embodiments, the assay is a cell-free assay, wherein themitochondrial NAD-dependent deacetylase is contacted with the testagent, the substrate (i.e., acetylated histone 4 peptide), and otherreaction components (e.g., NAD, buffers, and the like), and the activityof the mitochondrial NAD-dependent deacetylase determined. In theseembodiments, the mitochondrial NAD-dependent deacetylase may bepurified, but need not be. The mitochondrial NAD-dependent deacetylasemay be present in a cell extract; in an immunoprecipitate of a cellextract; or may be partially purified, e.g., at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, or more, purified, e.g., free of other macromolecules present inthe source of the mitochondrial NAD-dependent deacetylase. Themitochondrial NAD-dependent deacetylase may be recombinant, or may beisolated from a natural source, e.g., a mammalian cell or tissue thatnormally produced the enzyme.

Agents

The present invention further provides biologically active agentsidentified using a method of the instant invention. A biologicallyactive agent of the invention modulates a level or an activity of amitochondrial NAD-dependent deacetylase. Agents are useful to treatvarious disorders, including cancer, neurodegenerative disorders,metabolic disorders, and disorders associated with apoptosis.

In many embodiments, the agent is a small molecule, e.g., a smallorganic or inorganic compound having a molecular weight of more than 50and less than about 2,500 daltons. Agents may comprise functional groupsnecessary for structural interaction with proteins, particularlyhydrogen bonding, and may include at least an amine, carbonyl, hydroxylor carboxyl group, and may contain at least two of the functionalchemical groups. The agents may comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

In some embodiments, an active agent is a peptide. Suitable peptidesinclude peptides of from about 3 amino acids to about 50, from about 5to about 30, or from about 10 to about 25 amino acids in length. Apeptide of interest inhibits an enzymatic activity of mitochondrialNAD-dependent deacetylase.

Peptides can include naturally-occurring and non-naturally occurringamino acids. Peptides may comprise D-amino acids, a combination of D-and L-amino acids, and various “designer” amino acids (e.g., β-methylamino acids, Cα-methyl amino acids, and Nα-methyl amino acids, etc.) toconvey special properties to peptides. Additionally, peptide may be acyclic peptide. Peptides may include non-classical amino acids in orderto introduce particular conformational motifs. Any known non-classicalamino acid can be used. Non-classical amino acids include, but are notlimited to, 1,2,3,4-tetrahydroisoquinoline-3-carboxylate;(2S,3S)-methylphenylalanine, (2S,3R)-methyl-phenylalanine,(2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine;2-aminotetrahydronaphthalene-2-carboxylic acid;hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate; β-carboline (D andL); HIC (histidine isoquinoline carboxylic acid); and HIC (histidinecyclic urea). Amino acid analogs and peptidomimetics may be incorporatedinto a peptide to induce or favor specific secondary structures,including, but not limited to, LL-Acp(LL-3-amino-2-propenidone-6-carboxylic acid), a β-turn inducingdipeptide analog; β-sheet inducing analogs; β-turn inducing analogs;α-helix inducing analogs; γ-turn inducing analogs; Gly-Ala turn analog;amide bond isostere; tretrazol; and the like.

A peptide may be a depsipeptide, which may be a linear or a cyclicdepsipeptide. Kuisle et al. (1999) Tet. Letters 40:1203-1206.“Depsipeptides” are compounds containing a sequence of at least twoalpha-amino acids and at least one alpha-hydroxy carboxylic acid, whichare bound through at least one normal peptide link and ester links,derived from the hydroxy carboxylic acids, where “linear depsipeptides”may comprise rings formed through S—S bridges, or through an hydroxy ora mercapto group of an hydroxy-, or mercapto-amino acid and the carboxylgroup of another amino- or hydroxy-acid but do not comprise rings formedonly through peptide or ester links derived from hydroxy carboxylicacids. “Cyclic depsipeptides” are peptides containing at least one ringformed only through peptide or ester links, derived from hydroxycarboxylic acids.

Peptides may be cyclic or bicyclic. For example, the C-terminal carboxylgroup or a C-terminal ester can be induced to cyclize by internaldisplacement of the —OH or the ester (—OR) of the carboxyl group orester respectively with the N-terminal amino group to form a cyclicpeptide. For example, after synthesis and cleavage to give the peptideacid, the free acid is converted to an activated ester by an appropriatecarboxyl group activator such as dicyclohexylcarbodiimide (DCC) insolution, for example, in methylene chloride (CH₂Cl₂), dimethylformamide (DMF) mixtures. The cyclic peptide is then formed by internaldisplacement of the activated ester with the N-terminal amine. Internalcyclization as opposed to polymerization can be enhanced by use of verydilute solutions. Methods for making cyclic peptides are well known inthe art

The term “bicyclic” refers to a peptide in which there exists two ringclosures. The ring closures are formed by covalent linkages betweenamino acids in the peptide. A covalent linkage between two nonadjacentamino acids constitutes a ring closure, as does a second covalentlinkage between a pair of adjacent amino acids which are already linkedby a covalent peptide linkage. The covalent linkages forming the ringclosures may be amide linkages, i.e., the linkage formed between a freeamino on one amino acid and a free carboxyl of a second amino acid, orlinkages formed between the side chains or “R” groups of amino acids inthe peptides. Thus, bicyclic peptides may be “true” bicyclic peptides,i.e., peptides cyclized by the formation of a peptide bond between theN-terminus and the C-terminus of the peptide, or they may be“depsi-bicyclic” peptides, i.e., peptides in which the terminal aminoacids are covalently linked through their side chain moieties.

A desamino or descarboxy residue can be incorporated at the terminii ofthe peptide, so that there is no terminal amino or carboxyl group, todecrease susceptibility to proteases or to restrict the conformation ofthe peptide. C-terminal functional groups include amide, amide loweralkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, andthe lower ester derivatives thereof, and the pharmaceutically acceptablesalts thereof.

In addition to the foregoing N-terminal and C-terminal modifications, apeptide or peptidomimetic can be modified with or covalently coupled toone or more of a variety of hydrophilic polymers to increase solubilityand circulation half-life of the peptide. Suitable nonproteinaceoushydrophilic polymers for coupling to a peptide include, but are notlimited to, polyalkylethers as exemplified by polyethylene glycol andpolypropylene glycol, polylactic acid, polyglycolic acid,polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose andcellulose derivatives, dextran and dextran derivatives, etc. Generally,such hydrophilic polymers have an average molecular weight ranging fromabout 500 to about 100,000 daltons, from about 2,000 to about 40,000daltons, or from about 5,000 to about 20,000 daltons. The peptide can bederivatized with or coupled to such polymers using any of the methodsset forth in Zallipsky, S., Bioconjugate Chem., 6:150-165 (1995);Monfardini, C, et al., Bioconjugate Chem., 6:62-69 (1995); U.S. Pat.Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; 4,179,337 orWO 95/34326.

Another suitable agent for reducing an activity of a mitochondrialNAD-dependent deacetylase is a peptide aptamer. Peptide aptamers arepeptides or small polypeptides that act as dominant inhibitors ofprotein function. Peptide aptamers specifically bind to target proteins,blocking their function ability. Kolonin and Finley, PNAS (1998)95:14266-14271. Due to the highly selective nature of peptide aptamers,they may be used not only to target a specific protein, but also totarget specific functions of a given protein (e.g. a signalingfunction). Further, peptide aptamers may be expressed in a controlledfashion by use of promoters which regulate expression in a temporal,spatial or inducible manner. Peptide aptamers act dominantly; therefore,they can be used to analyze proteins for which loss-of-function mutantsare not available.

Peptide aptamers that bind with high affinity and specificity to atarget protein may be isolated by a variety of techniques known in theart. Peptide aptamers can be isolated from random peptide libraries byyeast two-hybrid screens (Xu et al., PNAS (1997) 94:12473-12478). Theycan also be isolated from phage libraries (Hoogenboom et al.,Immunotechnology (1998) 4:1-20) or chemically generatedpeptides/libraries.

Intracellularly expressed antibodies, or intrabodies, are single-chainantibody molecules designed to specifically bind and inactivate targetmolecules inside cells. Intrabodies have been used in cell assays and inwhole organisms. Chen et al., Hum. Gen. Ther. (1994) 5:595-601;Hassanzadeh et al., Febs Lett. (1998) 16(1, 2):75-80 and 81-86.Inducible expression vectors can be constructed with intrabodies thatreact specifically with mitochondrial NAD-dependent deacetylase protein.These vectors can be introduced into model organisms and studied in thesame manner as described above for aptamers.

In some of the invention, the active agent is an agent that modulates,and generally decreases or down regulates, the expression of the geneencoding mitochondrial NAD-dependent deacetylase in the host. Suchagents include, but are not limited to, antisense RNA, interfering RNA,ribozymes, and the like.

In some embodiments, the active agent is an interfering RNA (RNAi). RNAiincludes double-stranded RNA interference (dsRNAi). Use of RNAi toreduce a level of a particular mRNA and/or protein is based on theinterfering properties of double-stranded RNA derived from the codingregions of gene. In one example of this method, complementary sense andantisense RNAs derived from a substantial portion of the mitochondrialNAD-dependent deacetylase gene are synthesized in vitro. The resultingsense and antisense RNAs are annealed in an injection buffer, and thedouble-stranded RNA injected or otherwise introduced into the subject(such as in their food or by soaking in the buffer containing the RNA).See, e.g., WO99/32619. In another embodiment, dsRNA derived from amitochondrial NAD-dependent deacetylase gene is generated in vivo bysimultaneous expression of both sense and antisense RNA fromappropriately positioned promoters operably linked to mitochondrialNAD-dependent deacetylase coding sequences in both sense and antisenseorientations.

Antisense molecules can be used to down-regulate expression of the geneencoding mitochondrial NAD-dependent deacetylase in cells. Antisensecompounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression.

The anti-sense reagent may be antisense oligonucleotides (ODN),particularly synthetic ODN having chemical modifications from nativenucleic acids, or nucleic acid constructs that express such anti-sensemolecules as RNA. The antisense sequence is complementary to the mRNA ofthe targeted gene, and inhibits expression of the targeted geneproducts. Antisense molecules inhibit gene expression through variousmechanisms, e.g. by reducing the amount of mRNA available fortranslation, through activation of RNAse H, or steric hindrance. One ora combination of antisense molecules may be administered, where acombination may comprise multiple different sequences.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996), Nature Biotechnol. 14:840-844).

A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methodsknown in the art (see Wagner et al. (1993), supra, and Milligan et al.,supra.) Preferred oligonucleotides are chemically modified from thenative phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which modificationsalter the chemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′—NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The β-anomer of deoxyribose may be used, where the base isinverted with respect to the natural α-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

Exemplary modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Oligonucleotides having a morpholino backbone structure (Summerton, J.E. and Weller D. D., U.S. Pat. No. 5,034,506) or a peptide nucleic acid(PNA) backbone (P. E. Nielson, M. Egholm, R. H. Berg, O. Buchardt,Science 1991, 254: 1497) can also be used. Morpholino antisenseoligonucleotides are amply described in the literature. See, e.g.,Partridge et al. (1996) Antisense Nucl. Acid Drug Dev. 6:169-175; andSummerton (1999) Biochem. Biophys. Acta 1489:141-158.

As an alternative to anti-sense inhibitors, catalytic nucleic acidcompounds, e.g. ribozymes, anti-sense conjugates, etc. may be used toinhibit gene expression. Ribozymes may be synthesized in vitro andadministered to the patient, or may be encoded on an expression vector,from which the ribozyme is synthesized in the targeted cell (forexample, see International patent application WO 9523225, and Beigelmanet al. (1995), Nucl. Acids Res. 23:4434-42). Examples ofoligonucleotides with catalytic activity are described in WO 9506764.Conjugates of anti-sense ODN with a metal complex, e.g.terpyridylCu(II), capable of mediating mRNA hydrolysis are described inBashkin et al. (1995), Appl. Biochem. Biotechnol. 54:43-56.

Formulations, Dosages, and Routes of Administration

The invention provides formulations, including pharmaceuticalformulations, comprising an agent that reduces a level and/or anactivity of mitochondrial NAD-dependent deacetylase. In general, aformulation comprises an effective amount of an agent that reduces alevel and/or an activity of mitochondrial NAD-dependent deacetylase. An“effective amount” means a dosage sufficient to produce a desiredresult, e.g., a reduction in a level and/or an activity of mitochondrialNAD-dependent deacetylase, a reduction in histone deacetylation; and thelike. Generally, the desired result is at least a reduction a leveland/or an activity of mitochondrial NAD-dependent deacetylase ascompared to a control.

Formulations

In the subject methods, the active agent(s) may be administered to thehost using any convenient means capable of resulting in the desiredreduction in a level and/or an activity of mitochondrial NAD-dependentdeacetylase. Thus, the agent can be incorporated into a variety offormulations for therapeutic administration. More particularly, theagents of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants and aerosols.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

Other modes of administration will also find use with the subjectinvention. For instance, an agent of the invention can be formulated insuppositories and, in some cases, aerosol and intranasal compositions.For suppositories, the vehicle composition will include traditionalbinders and carriers such as, polyalkylene glycols, or triglycerides.Such suppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10% (w/w), preferablyabout 1% to about 2%.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

An agent of the invention can be administered as injectables. Typically,injectable compositions are prepared as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid vehiclesprior to injection may also be prepared. The preparation may also beemulsified or the active ingredient encapsulated in liposome vehicles.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985. The composition or formulation to be administered will,in any event, contain a quantity of the agent adequate to achieve thedesired state in the subject being treated.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Dosages

Although the dosage used will vary depending on the clinical goals to beachieved, a suitable dosage range is one which provides up to about 1 μgto about 1,000 μg or about 10,000 μg of an agent that reduces a leveland/or an activity of mitochondrial NAD-dependent deacetylase can beadministered in a single dose. Alternatively, a target dosage of anagent that reduces a level and/or an activity of mitochondrialNAD-dependent deacetylase can be considered to be about in the range ofabout 0.1-1000 μM, about 0.5-500 μM, about 1-100 μM, or about 5-50 μM ina sample of host blood drawn within the first 24-48 hours afteradministration of the agent.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Preferred dosages for agiven compound are readily determinable by those of skill in the art bya variety of means.

Routes of Administration

An agent that reduces a level and/or an activity of mitochondrialNAD-dependent deacetylase is administered to an individual using anyavailable method and route suitable for drug delivery, including in vivoand ex vivo methods, as well as systemic and localized routes ofadministration.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, intratumoral,subcutaneous, intradermal, topical application, intravenous, rectal,nasal, oral and other parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe agent and/or the desired effect. The composition can be administeredin a single dose or in multiple doses.

The agent can be administered to a host using any available conventionalmethods and routes suitable for delivery of conventional drugs,including systemic or localized routes. In general, routes ofadministration contemplated by the invention include, but are notnecessarily limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, and intravenous routes, i.e., any route of administrationother than through the alimentary canal. Parenteral administration canbe carried to effect systemic or local delivery of the agent. Wheresystemic delivery is desired, administration typically involves invasiveor systemically absorbed topical or mucosal administration ofpharmaceutical preparations.

The agent can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

Methods of administration of the agent through the skin or mucosainclude, but are not necessarily limited to, topical application of asuitable pharmaceutical preparation, transdermal transmission, injectionand epidermal administration. For transdermal transmission, absorptionpromoters or iontophoresis are suitable methods. Iontophoretictransmission may be accomplished using commercially available “patches”which deliver their product continuously via electric pulses throughunbroken skin for periods of several days or more.

By treatment is meant at least an amelioration of the symptomsassociated with the pathological condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thepathological condition being treated, such as an allergichypersensitivity. As such, treatment also includes situations where thepathological condition, or at least symptoms associated therewith, arecompletely inhibited, e.g. prevented from happening, or stopped, e.g.terminated, such that the host no longer suffers from the pathologicalcondition, or at least the symptoms that characterize the pathologicalcondition.

A variety of hosts (wherein the term “host” is used interchangeablyherein with the terms “subject” and “patient”) are treatable accordingto the subject methods. Generally such hosts are “mammals” or“mammalian,” where these terms are used broadly to describe organismswhich are within the class mammalia, including the orders carnivore(e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), andprimates (e.g., humans, chimpanzees, and monkeys). In many embodiments,the hosts will be humans.

Kits with unit doses of the active agent, e.g. in oral or injectabledoses, are provided. In such kits, in addition to the containerscontaining the unit doses will be an informational package insertdescribing the use and attendant benefits of the drugs in treatingpathological condition of interest. Preferred compounds and unit dosesare those described herein above.

Therapeutic Methods

The invention further provides methods of treating various disorders, bymodulating a level or an activity of a mitochondrial NAD-dependentdeacetylase. The methods generally involve administering to anindividual in need thereof an effective amount of an agent thatmodulates a level or an activity of a mitochondrial NAD-dependentdeacetylase. In some embodiments, the methods involve decreasing a levelor activity of a mitochondrial NAD-dependent deacetylase. In otherembodiments, the methods involve increasing a level or activity of amitochondrial NAD-dependent deacetylase.

Increasing a level or activity of a mitochondrial NAD-dependentdeacetylase provides a protective effect against apoptosis. In someembodiments, an effective amount of an agent that increases a level ofactivity of a mitochondrial NAD-dependent deactylase is an amount thatis effective to decrease apoptosis by at least about 10%, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, or at leastabout 90% or more, when compared to the level of apoptosis in anindividual not treated with the agent.

Decreasing a level or activity of a mitochondrial NAD-dependentdeacetylase increases apoptosis. Increasing apoptosis is desirable inthe context of reducing unwanted cellular proliferation. In someembodiments, an effective amount of an agent that decreases a level ofactivity of a mitochondrial NAD-dependent deactylase is an amount thatis effective to increase apoptosis by at least about 10%, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, or at leastabout 90% or more, when compared to the level of apoptosis in anindividual not treated with the agent.

Disorders amenable to treatment using a method according to theinvention are disorders related to, associated with, or caused (directlyor indirectly) by mitochondrial malfunction or dysfunction. Disordersamenable to treatment using a method according to the invention includecancer; neurodegenerative disorders; metabolic disorders;ischemia-reperfusion injury; and disorders associated with apoptosis orcell death.

Indications which can be treated using the methods of the invention forreducing apoptosis or cell death in a eukaryotic cell, include, but arenot limited to, cell death or apoptosis associated with Alzheimer'sdisease, Parkinson's disease, rheumatoid arthritis, septic shock,sepsis, stroke, central nervous system inflammation, osteoporosis,ischemia (e.g., resulting from stroke or myocardial infarction),reperfusion injury, cell death associated with cardiovascular disease,polycystic kidney disease, cell death of endothelial cells incardiovascular disease, degenerative liver disease, multiple sclerosis,amyotropic lateral sclerosis, cerebellar degeneration, ischemic injury,cerebral infarction, myocardial infarction, myelodysplastic syndromes,aplastic anemia, male pattern baldness, and head injury damage. Alsoincluded are any hypoxic or anoxic conditions, e.g., conditions relatingto or resulting from ischemia, myocardial infarction, cerebralinfarction, stroke, bypass heart surgery, organ transplantation,neuronal damage, and the like.

Cell death-related indications which can be treated using methods of theinvention for activating apoptosis or cell death include, but are notlimited to, undesired, excessive, or uncontrolled cellularproliferation, including, for example, neoplastic cells; as well as anyundesired cell or cell type in which induction of cell death is desired,e.g., virus-infected cells and self-reactive immune cells. The methodsmay be used to treat follicular lymphomas, carcinomas associated withp53 mutations; autoimmune disorders, such as, for example, systemiclupus erythematosus (SLE), immune-mediated glomerulonephritis;hormone-dependent tumors, such as, for example, breast cancer, prostatecancer and ovary cancer; and viral infections, such as, for example,herpesviruses, poxviruses and adenoviruses.

Whether a therapeutic method of the invention is effective in modulatingcell death/apoptosis can be determined using any known assay. Cell deathcan be measured using any known method, and is generally measured usingany of a variety of known methods for measuring cell viability. Suchassays are generally based on entry into the cell of a detectablecompound (or a compound that becomes detectable upon interacting with,or being acted on by, an intracellular component) that would normally beexcluded from a normal, living cell by its intact cell membrane. Suchcompounds include substrates for intracellular enzymes, including, butnot limited to, a fluorescent substrate for esterase; dyes that areexcluded from living cell, including, but not limited to, trypan blue;and DNA-binding compounds, including, but not limited to, an ethidiumcompound such as ethidium bromide and ethidium homodimer, and propidiumiodide.

Apoptosis can be assayed using any known method. Assays can be conductedon cell populations or an individual cell, and include morphologicalassays and biochemical assays. A non-limiting example of a method ofdetermining the level of apoptosis in a cell population is TUNEL(TdT-mediated dUTP nick-end labeling) labeling of the 3′-OH free end ofDNA fragments produced during apoptosis (Gavrieli et al. (1992) J. CellBiol. 119:493). The TUNEL method consists of catalytically adding anucleotide, which has been conjugated to a chromogen system or a to afluorescent tag, to the 3′-OH end of the 180-bp (base pair) oligomer DNAfragments in order to detect the fragments. The presence of a DNA ladderof 180-bp oligomers is indicative of apoptosis. Procedures to detectcell death based on the TUNEL method are available commercially, e.g.,from Boehringer Mannheim (Cell Death Kit) and Oncor (Apoptag Plus).Another marker that is currently available is annexin, sold under thetrademark APOPTEST™. This marker is used in the “Apoptosis DetectionKit,” which is also commercially available, e.g., from R&D Systems.During apoptosis, a cell membrane's phospholipid asymmetry changes suchthat the phospholipids are exposed on the outer membrane. Annexins are ahomologous group of proteins that bind phospholipids in the presence ofcalcium. A second reagent, propidium iodide (PI), is a DNA bindingfluorochrome. When a cell population is exposed to both reagents,apoptotic cells stain positive for annexin and negative for PI, necroticcells stain positive for both, live cells stain negative for both. Othermethods of testing for apoptosis are known in the art and can be used,including, e.g., the method disclosed in U.S. Pat. No. 6,048,703.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric. Standard abbreviations may beused, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s,second(s); min, minute(s); hr, hour(s); and the like.

Example 1 Characterization of a Mitochondrial NAD-Dependent HistoneDeacetylase Experimental Procedures Plasmid Construction

Plasmids expressing hSIRT3 were constructed by polymerase chain reaction(PCR) amplification of the hSIRT3 coding sequence using primerscontaining EcoRI sites and pCR2.1-SIRT3 as a template. Amplifiedsequences were digested with EcoRI and cloned into a modifiedpcDNA3.1+vector (Invitrogen, Carlsbad, Calif.) to yield a C-terminallyFLAG-tagged hSIRT3. hSIRT3Δ1-25-FLAG was constructed by using modifiedN-terminal PCR primers introducing EcorI sites and a methionine startcodon before amino acid 26 of the wild-type protein. Site-directedmutagenesis (QuikChange™ Mutagenesis Kit, Stratagene, La Jolla, Calif.)was used for construction of hSIRT3N229A-FLAG, hSIRT3H248Y-FLAG,hSIRT3R7/13G-FLAG, hSIRT3R17/21G-FLAG, hSIRT3R7/13/17/21G-FLAG,hSIRT3R7/13Q-FLAG, hSIRT3R17/21Q-FLAG, hSIRT3R7/13/17/21Q-FLAG,hSIRT3L12P/R13P-FLAG and hSIRT3R99/100G-FLAG. All constructs wereverified by DNA sequencing. pSu9-DHFR was provided by J. Brix and N.Pfanner (Institut fuer Biochemie und Molekularbiologie, Freiburg,Germany).

GFP Fusion Constructs

To generate fusion proteins of GFP with wild-type hSIRT3 or with aminoacid 26-399 of hSIRT3, corresponding coding sequences were PCR amplifiedand cloned into pEGFP-N1 (Clontech, Palo Alto, Calif.).

Cell Culture and Transfection

HEK293T and HeLa cells were cultured in DMEM supplemented with 10% FCS,2 mM L-glutamine, 100 U Penicillin and 100 μg Streptomycin per ml andgrown in 5% CO₂ at 37° C. Calcium phosphate transfection was used totransfect HEK293T cells (19). HeLa cells were transfected withLipofectamine (Life Technologies, Rockville, Md.).

Immunoblot Analysis

Antibodies used for immunoblotting included anti-mtHsp70 (Clone JG1,Affinity Bioreagents, Golden, Colo.), anti-Hsp60 (Clone 4B9/89, AffinityBioreagents, Golden, Colo.), anti-Hsp90α (StressGen, Victoria, Canada),anti-cytochrome c oxidase subunit IV (Clone 20E8-C12, Molecular Probes,Eugene, Oreg.), anti-FLAG M2 (Sigma, St. Louis, Mo.), anti-cytochrome c(Clone 7H8.2C12, Pharmingen, San Diego, Calif.). hSIRT3 antisera wereraised in rabbits against a C-terminal peptide(H₂N-DLVQRETGKLDGPDK-COOH; SEQ ID NO:05). Western blots were revealedwith enhanced chemiluminescence (Amersham Pharmacia, Piscataway, N.J.).Membranes were either nitrocellulose (Hybond ECL, Amersham Pharmacia,Piscataway, N.J.) or PVDF (Immun-Blot™, Bio-Rad, Hercules, Calif.).

Immunofluorescence and Confocal Microscopy

HeLa cells grown on coverslips were incubated for 45 min with 30 nMMitoTracker (CMXRos, Molecular Probes, Inc., Eugene, Oreg.) in DMEM minat 37° C., transferred to fresh DMEM and further incubated for 60 min.Cells on coverslips were rinsed in phosphate-buffered saline (PBS),fixed in 3.7% formaldehyde/PBS for 30 min, washed again in PBS andmounted. Images were acquired on a BioRad Radiance 2000 laser scanningmicroscope equipped with an Olympus BX60 microscope using an OlympusPlanApo 60x/1.40 oil objective. Excitation laser line was 488 nm forenhanced green fluorescent protein (eGFP) and 578 nm for MitoTracker.

Preparation of Subcellular Fractions

Subcellular fractionation was performed according to publishedprocedures with minor modifications (20,21). All steps were performed at4° C. In brief, cells were homogenized in ice-cold buffer A (250 mMsucrose, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mMdithiotreithol, 0.1 mM phenylmethylsulfonyl fluoride, 20 mM HEPES-KOH,pH 7.5) and homogenized in a Dounce homogenizer (Wheaton, Millville,N.J.). Homogenization was checked by phase-contrast microscopy. Thehomogenate was centrifuged twice at 800×g to remove nuclei and unbrokencells. Mitochondria were sedimented by centrifugation at 7,000×g for 15min at 4° C., washed twice with buffer A and resuspended in TXIP-1buffer (1% Triton X-100 (v/v), 150 mM NaCl, 0.5 mM EDTA, 50 mM Tris-HCl,pH 7.4) supplemented with protease inhibitors. Postmitochondrialsupernatants were fractionated by ultracentrifugation at 100,000×g for30 min at 4° C. The supernatant constituting the cytosolic S-100fraction was removed and the pellet was resuspended in TXIP-1 buffer.Protein concentrations of the fractions were determined (DC ProteinAssay, Bio-Rad, Hercules, Calif.) and equal amounts of each fractionwere separated by SDS-PAGE and blotted to nitrocellulose.

Isolation of Mitochondria from Mammalian Cells

Mitochondria were isolated by differential centrifugation according topublished procedures (21). After several washes in SEM buffer (250 mMsucrose, 1 mM EDTA, 10 mM MOPS-KOH, pH 7.2), mitochondria wereresuspended in SEM buffer. To further purify mitochondria, a crudemitochondrial fraction was layered on a discontinuous sucrose gradient(1-1.5 M) in T₁₀E₁ buffer (1 mM EDTA, 10 mM Tris-HCl, pH 7.5). Aftercentrifugation for 20 min at 60,000×g at 4° C., mitochondria wererecovered from the 1.0 M/1.5 M interface, carefully adjusted to 250 mMsucrose and washed twice in SEM buffer.

Immunoprecipitation

Cells or isolated mitochondria were lysed in ice-cold TXIP-1 buffercontaining either PMSF or protease inhibitor cocktail (Roche,Indianapolis, Ind.). Lysates were centrifuged at 16,000×g for 5 min at4° C. and Anti-FLAG monoclonal M2 antibody (Sigma, St. Louis, Mo.)covalently coupled to agarose was added. Samples were incubated at 4° C.for 12 hrs, centrifuged and washed 4 times in TXIP-1 buffer. For thedeacetylation assays, the fourth wash was carried out in SIRTdeacetylase buffer (4 mM MgCl2, 0.2 mM dithiothreitol, 50 mM Tris-HCl,pH 9.0)

Import of Radiolabeled Proteins into Isolated Mitochondria

Import into isolated mitochondria was carried out as previously reported(22). Proteins were synthesized in the presence of [³⁵S]-methionine bycoupled transcription-translation in reticulocyte lysate (Promega,Madison, Wis.) (23). In vitro translation reactions were centrifuged at108,000×g, 2° C. for 15 min and adjusted to 250 mM sucrose. Importreactions contained 5% (v/v) reticulocyte lysate in import buffer (3%(w/v) fatty-acid free bovine serum albumin (BSA), 250 mM sucrose, 80 mMKCl, 5 mM MgCl₂, 2 mM KH₂PO₄, 5 mM L-methionine, 10 mM3-[N-morpholino]propanesulfonic acid-KOH, pH 7.2). In each importreaction, 50 μg of freshly isolated mammalian mitochondria were mixedwith radiolabeled proteins and incubated at 30° C. ATP (2 mM) and sodiumsuccinate (10 mM) were added to maintain coupling of isolatedmitochondria. Import was stopped by addition of valinomycin (1 μM) andtransfer to 0° C.

Where indicated, samples were treated with proteinase K (50 μg/ml) for10 min on ice. Protease treatment was stopped by addition of 2 mMphenylmethylsulfonyl fluoride (PMSF). Mitochondria were reisolated bycentrifugation at 10,000×g for 5 min at 4° C., washed in SEM buffer andrecentrifuged as above. Mitochondrial pellets were resuspended in SDSsample buffer containing dithiothreitol (DTT) and heated to 95° C. for 5min. Samples were subjected to SDS-PAGE. Dried gels were exposed toBiomax MR film (Kodak, Rochester, N.Y.) at −70° C. and analyzed on aFuji FUJIX BAS 1000 phosphorimager. Where indicated, mitochondrialtransmembrane potential was disrupted by blocking of complex III of therespiratory chain (Antimycin, 8 μM), blocking of the F₀/F₁-ATPase(Oligomycin, 20 μM) and potassium flux (Valinomycin, 1 μM).

Swelling experiments were performed according to published protocols(24). Mitochondria were isolated from hSIRT3-FLAG transfected HEK293Tcells, washed and treated with proteinase K (150 μg/ml) to removenonimported protein. Mitochondria were reisolated at 10 000×g for 5 min,washed with SEM buffer and recentrifuged. Mitochondrial pellets wereresuspended in SM buffer (250 mM sucrose, 10 mM MOPS-KOH, pH 7.2) andswollen by diluting them tenfold dilution into M buffer (10 mM MOPS-KOH,pH 7.2) and incubation on ice for 15 min. Mitoplasts and non-swollenmitochondria were treated with proteinase K (150 μg/ml) for 10 min at 0°C. Protease digestion was stopped by addition of 2 mM PMSF andmitoplasts and mitochondria were reisolated by centrifugation, washedand lysed in sample buffer. Samples were separated by SDS-PAGE andblotted onto PVDF membrane. Radiolabeled proteins were detected byautoradiography.

Fractionation of Mitochondrial Proteins by Alkaline Treatment

These experiments were performed using published protocols (25,26). Inbrief, washed mitochondrial pellets were resuspended in freshly prepared0.1 M sodium carbonate, pH 11.5, and incubated at 0° C. for 30 min.Mitochondrial membranes were sedimented by ultracentrifugation at100,000×g for 30 min at 4° C. The pellet was resuspended in SDS samplebuffer and proteins in the supernatant were concentrated bytrichloracetate precipitation and finally resuspended in sample buffer.

In Vitro Deacetylase Assay

Deacetylase assays were performed in a total volume of 100 μl SIRTdeacetylase buffer (4 mM MgCl₂, 0.2 mM dithiothreitol, 50 mM Tris-HCl,pH 9.0) containing immunoprecipitated proteins or mitochondrial lysatesand a peptide corresponding to the first the first 22 amino acids ofhistone 4 chemically acetylated in vitro (27). Where indicated, 1 mMNAD, 5 mM nicotinamide (both from Sigma, St. Louis, Mo.) or 400 nM TSA(WAKO, Richmond, Va.) were added. Deacetylation reactions were stoppedafter 2 hours of incubation at room temperature by adding 25 μl stopsolution (0.1 M HCl, 0.16 M acetic acid). Released acetate was extractedinto 500 μl ethyl acetate and samples were vigorously shaken for 15minutes. After centrifugation for 5 minutes, 400 μl of the ethyl acetatefraction was mixed with 5 ml scintillation fluid (Packard, Meriden,Conn.) and the released radioactivity was measured using a liquidscintillation counter.

Mitochondrial Processing Peptidase Cleavage Assay

Purified recombinant yeast MPP (28) was obtained from G. Isaya (MayoClinic and Foundation, Rochester, Minn.). Cleavage of radiolabeled invitro translated proteins was carried out in reaction buffer (1 mMdithiothreitol, 1 mM MnCl₂, 10 mM Hepes-KOH, pH 7.4). Purified MPP orreaction buffer was added to each sample followed by incubation at 27°C. for 45 min. Reactions were stopped by addition of SDS sample bufferand boiling at 95° C. for 5 min. Samples were separated by SDS-PAGE andanalyzed by phosphorimaging.

Results Mitochondria Contain Sir2-Like Deacetylase Activity.

A systematic survey of subcellular fractions for the presence of histonedeacetylase activities led to the detection of a deacetylase activity inhuman mitochondrial fractions prepared from HEK293 T cells (FIG. 1A).This activity was strictly dependent on the presence of NAD and wassuppressed by nicotinamide (Vitamin B3), a product of NAD hydrolysis(29-31) reported to inhibit Sir2-like proteins (32) (FIG. 1A). Incontrast, trichostatin A (TSA), a specific inhibitor of class I andclass II deacetylases, had no effect on the deacetylase activity presentin mitochondria (FIG. 1A). Under the same conditions, TSA treatment ledto a significant inhibition of the activity of a prototypic class IIHDAC, HDAC6. The observed NAD-dependent deacetylase activity sensitiveto inhibition by nicotinamide but not by TSA indicated the presence ofSir2-like class III protein deacetylases in mitochondria.

hSIRT3 Mediates NAD-Dependent Deacetylase Activity in the Mitochondria.

Transfection of each hSIRT cDNA in mammalian cells followed byimmunoprecipitation and incubation with a histone H4 peptide substrateshowed that hSIRT1, 2 and 3 exhibited bona fide NAD-dependentdeacetylase activity while hSIRT4, 5, 6 and 7 showed no detectableactivity. To determine which hSIRT protein contributed to themitochondrial activity, expression vectors for hSIRT1, hSIRT2 and hSIRT3(epitope-tagged with FLAG at the C-terminus), or a control vector, weretransfected into HEK293T cells. Cells were harvested and half of thepreparation was used to prepare a whole cell lysate while the other halfwas used to isolate and purify mitochondria (mitochondrial lysate).hSIRT proteins were immunoprecipitated with anti-FLAG antibodies fromwhole cell or from mitochondria lysates and tested by western blottingfor the presence of the protein. All three proteins were expressed anddetected in whole cell lysates (FIG. 1B). Interestingly, two forms ofhSIRT3 were detected, a 44 KDa product of the expected size given thecDNA sequence (predicted molecular weight=43.6 KDa) and a smaller, 28KDa product (FIG. 1B).). In contrast, anti-FLAG immunoprecipitatesprepared from mitochondria, showed only the presence of hSIRT3 (28 KDaproduct) but not of hSIRT1 and 2 (FIG. 1B). Testing of the sameimmunoprecipitates for enzymatic activity yielded the same results.While all three hSIRTs showed robust NAD-dependent enzymatic activityafter immunoprecipitation from whole cell lysates (FIG. 1C), onlyanti-FLAG immunoprecipitates from cells transfected with hSIRT3 showedmitochondrial deacetylase activity (FIG. 1D). These results areconsistent with the model that hSIRT3 can target mitochondria andmediate NAD-dependent deacetylase activity within that subcellularcompartment.

When total mitochondrial lysates prepared from cells transfected withhSIRT3 were analyzed in the same in vitro deacetylase assay, an increasein NAD-dependent deacetylase activity was observed in comparison tocells transfected with a control plasmid (FIG. 1E). The mitochondriallysates overexpressing hSIRT3 exhibited the same properties asuntransfected mitochondrial lysates in terms of sensitivity tonicotinamide and TSA. In contrast, transfection of two catalyticallyinactive mutants, hSIRT3-N229A and hSIRT3-H248Y, had no effect on theactivity of the lysates (FIG. 1E). These two mutants were designed byhomology to similar mutations reported to abrogate the activity ofSir2-like proteins. Both mutants were shown in separate experiments tobe catalytically inactive in whole cell lysates. Importantly, bothmutants were efficient targeted to mitochondria, were equally wellexpressed after transfection and were processed to the smaller 28 KDaproduct as wild type hSIRT3 (FIG. 1F). These observations are consistentwith the selective targeting of exogenous hSIRT3 to mitochondria.

FIG. 1A: Mitochondria contain Sir2-like deacetylase activity.Mitochondrial lysates were prepared from HEK293T cells and proteincontent was determined. Equal amounts of lysate were assayed fordeacetylase activity on a histone H4 peptide in either the presence orabsence of NAD (1 mM) or in combination with nicotinamide (5 mM) or TSA(400 nM). Samples were incubated for 2 hrs at 25° C. Released acetatewas quantitated as described in Materials and Methods. Representativeresults are shown.

FIG. 1B: In vitro deacetylase activity assay. hSIRT proteins wereimmunoprecipitated from whole cell lysate from transfected HEK293T cellsusing anti-FLAG antibodies. Immunoprecipitated proteins were assayed inthe presence or absence of NAD (1 mM).

FIG. 1C: Purified mitochondria from HEK293T cells transfected with hSIRTproteins were lysed and FLAG-tagged proteins were immunoprecipitated andanalyzed for in vitro deacetylase activity.

FIG. 1D: Western blot analysis of the immunoprecipitates obtained fromwhole cell lysate (upper panel) or purified mitochondria (lower panel).50% of the immunoprecipitate used in the deacetylase assay was detectedusing anti-FLAG M2 antibodies.

FIG. 1E: Transfection of hSIRT3 increases the NAD-dependent deacetylaseactivity of mitochondria. Mitochondria were isolated from HEK293T cellstransfected with hSIRT3-FLAG, hSIRT3N229A-FLAG, hSIRT3H248Y-FLAG orcontrol vector (pFLAG) and lysed in TXIP-1 buffer. In vitro deacetylaseactivities of equal amounts of mitochondrial lysate are shown.

FIG. 1F: Mitochondria were analyzed for the presence of hSIRT3 wild-typeand mutants by western blotting.

Endogenous and Exogenous hSIRT3 are Mitochondrial Proteins.

To further determine the subcellular localization of hSIRT3 in cells, afusion protein with green fluorescent protein was generated(hSIRT3-GFP). Confocal laser scanning microscopy of HeLa cellstransfected with hSIRT3-GFP revealed that it localized exclusively tocytoplasmic substructures consistent with mitochondria. This predictionwas verified by costaining with a mitochondria-specific dye, MitoTrackerred, which showed total overlapping of the two signals. This experimentindicated that hSIRT3 exclusively localizes to mitochondria.

This observation was further verified using cell fractionationexperiments. Cells transfected with hSIRT3-FLAG were used to preparesubcellular fractions according to established protocols (21). Equalamounts of protein from each subcellular fraction were subjected toSDS-PAGE and immunoblotting using an antibody specific for theC-terminal FLAG-epitope. hSIRT3-FLAG and cytochrome c were only bedetected within the heavy membrane fraction representing mitochondria(HM, FIG. 2A). Two FLAG-reactive bands were detected within themitochondrial fraction as discussed above (see FIG. 2A). Immunoblottingof subfractions prepared from untransfected cell confirmed that bothbands were specific for hSIRT3-FLAG.

Endogenous Mitochondrial hSIRT3 Protein has NAD-Dependent DeacetylaseActivity.

To examine the subcellular localization of endogenous hSIRT3, a specificantiserum was raised against the a peptide corresponding the last 15amino acids of hSIRT3 (N-DLVQRETGKLDGPDK-C; SEQ ID NO:06). Thisantiserum recognized two peptides of ˜44 and ˜28 KDa in mitochondriafraction while the preimmune antiserum obtained from the same rabbit wasunreactive to these proteins (FIG. 2B). These two bands corresponded insize to the ˜44 and 28 kDa fragment detected after transfection of theFLAG-tagged hSIRT3. Immunoprecipitation of mitochondria fraction withthis antiserum showed the presence of a specific NAD-dependentdeacetylase activity which was not present with the preimmune serum orwith Protein G sepharose alone (FIG. 2C). These experiments demonstratethat endogenous hSIRT3 is located in the mitochondria and is associatedwith NAD-dependent deacetylase activity in that compartment.

FIG. 2A: Subcellular fractionation of HEK293T cells transfected withhSIRT3-FLAG were homogenized and fractionated by differentialcentrifugation. Equal amounts (30 μg) of HM (heavy membranes), LM (lightmembranes) and S-100 (cytosolic proteins) fraction were analyzed byimmunoblotting. hSIRT3-FLAG was revealed by detection with monoclonal M2anti-FLAG antibodies. Two hSIRT3-FLAG specific forms (asterisks) weredetected. Nitrocellulose membranes were stripped and reprobed withantibodies against cytochrome c (cyt c) and Hsp90α.

FIG. 2 B: Detection of endogenous hSIRT3 protein in mitochondriallysates. Mitochondria were prepared from HEK293T cells. Lysates wereanalyzed by western blotting using a polyclonal rabbit hSIRT3 antiserum(35 μg/ml) or a preimmune serum (35 μg/ml) obtained from the samerabbit.

FIG. 2C: Endogenous hSIRT3 protein has NAD-dependent deacetylaseactivity in vitro. hSIRT3 was immunoprecipitated from HEK293T cellslysed in TXIP-1 buffer using hSIRT3 antiserum (0.35 mg/ml), preimmuneserum (0.35 mg/ml) or protein G sepharose. Equal amounts ofimmunoprecipitate were analyzed for in vitro deacetylase activity.

The N-Terminus of hSIRT3 is Required for Mitochondrial Import.

Mitochondrial targeting signals frequently contain an amphipatic α-helixand tend to contain positively charged, hydrophobic and hydroxylatedamino acid residues (15-18). Secondary structure prediction of hSIRT3revealed that an N-terminal peptide corresponding to residues 1-25 has ahigh probability to contain an amphipatic alpha-helix (34,35) (FIG. 3,middle panel). When plotted as a helical wheel (FIG. 3, right panel)residues 4 to 21, showed a cluster of positively charged arginineresidues on one side of the helix opposed by hydrophobic residues on theother side, a typical feature of mitochondrial presequences (reviewed in(36)). To test the importance of this putative alpha-helix in hSIRT3mitochondrial import, amino acid residues 1 to 25 were deleted fromhSIRT3 and fused it to GFP (hSIRT3Δ1-25-GFP). Expression of thisconstruct in HeLa cells showed pancellular distribution. No significantcolocalization between the fusion protein and MitoTracker-stainedmitochondria could be observed. This localization was in sharp contrastto the subcellular localization observed after expression of full-lengthhSIRT3 protein fused to GFP and indicated that the N-terminal 25 aminoacids of hSIRT3 are necessary for mitochondrial targeting.

FIG. 3. The N-terminal region of hSIRT3 is required for mitochondrialtargeting. Schematic diagram of hSIRT3. The hatched box illustrates theregion involved in mitochondrial targeting (left panel). Parts of theN-terminal region show a high probability to form an amphiphatic α helix(middle panel). Illustration of residues 4 to 21 as a helical wheel plotreveals a cluster of basic amino acids (black) on one side of theputative helix (right panel).

To further define the requirement for mitochondrial import of hSIRT3,cell-free mitochondrial in vitro import assays were used. Similar assayshave been used to elucidate the import requirements of a variety ofmitochondrial proteins. [³⁵S]-labeled hSIRT3 or hSIRT3Δ1-25 proteinswere synthesized in rabbit reticulocyte lysates and incubated withisolated mammalian mitochondria at 30° C. for 2, 5 or 15 minutes in thepresence of succinate and ATP. Mitochondria were reisolated from themixture by centrifugation and cosedimenting proteins were analyzed bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)analysis followed by autoradiography. A time-dependent accumulation ofhSIRT3, but not of hSIRT3Δ1-25, into mitochondria was observed (FIGS. 4Aand B). The import of SIRT across the mitochondrial membrane wasdependent on the mitochondrial transmembrane potential (ΔΨm) sinceimport was inhibited in the presence of antimycin (8 μM), oligomycin,(20 μM) and valinomycin (1 μM). (FIG. 4A, lane 4).

When the proteinase K digestion performed at the end of the importreaction was omitted, it was noted that both hSIRT3 and hSIRT3Δ1-25could bind to the outer surface of mitochondria in vitro, indicatingthat adhesion to mitochondria was not dependent on the N-terminal 25amino acids of hSIRT3. To exclude the possibility that proteins hadaggregated and cosedimented nonspecifically, similar experiments werecarried out in the absence of mitochondria, but no unspecificsedimentation occurred.

To further define the sequence and structural requirements necessary forimport of hSIRT3 into mitochondria, a series of point mutations in thefirst 25 amino acids was generated. We used two different strategies.First, we disrupted the α helix by introducing 2 prolines at position 12and 13. Second, we modified the charge of the amphipathic helix byreplacing arginine residues with glycine or glutamine. The polar butuncharged glutamine residues were predicted to preserve the α helicalconformation while changing the amphipathic character of the α helix. Tostudy the import efficiency, mutants and wild type hSIRT3 weresynthesized in rabbit reticulocyte lysates in the presence of[³⁵S]-methionine and assayed using the in vitro import assay describedabove. Mutation of R7 and R13 in either glycine or glutamine resulted ina loss of mitochondrial import. In contrast, mutation of R17 and R21reduced import by ˜50% (FIG. 4C). When all arginine residues weremutated into glutamine or glycine import efficiency was even furtherreduced. Disruption of the putative helical structure by two prolinesled to a loss in mitochondrial import similar to the R7/13G mutant.These results demonstrate the importance of the positively chargedresidues and of the α helical structure of region 1-25 in hSIRT3 for itsimport into mitochondria.

FIG. 4. Mitochondrial import of hSIRT3. FIG. 4A, [³⁵S]-labeledhSIRT3-FLAG or hSIRT3Δ1-25-FLAG synthesized in rabbit reticulocytelysate was imported into isolated mammalian mitochondria at 30° C. Toassay import in the absence of Δψm (lane 4), valinomycin (1 μM),antimycin (8 μM) and oligomycin (20 μM) were added to mitochondria 5 minprior to the addition of proteins. At indicated timepoints, furtherimport was stopped by dissipating Δψm (addition of 1 μM valinomycin) andincubation at 0° C. Samples from each timepoint were treated withproteinase K (50 μg/ml) for 10 min at 0° C. to remove nonimportedproteins. After reisolation of mitochondria and SDS-PAGE, the amounts ofimported proteins were quantified by phosphorimaging. FIG. 4B,Quantitation of imported protein by phosphorimaging. FIG. 4C, Schematicillustration of mutants used to address the effects of charged residuesand conformation on hSIRT3 import. FIG. 4D, [³⁵S]-labeled hSIRT3wild-type or mutants were imported into isolated mitochondria for 20 minat 30° C. Import was stopped as described above and nonimported proteinswere removed by proteinase K treatment. Reisolated and washedmitochondria were lysed in SDS sample buffer and analyzed by SDS-PAGE.Standards representing 50% of the input used in the individual importreactions were loaded adjacent to each import sample. FIG. 4E, Importefficiency of individual hSIRT3 mutants was quantitated in relation totheir standards by phosphorimaging. The import efficiency of hSIRT3 wasset to 100%.

hSIRT3 is a Mitochondrial Matrix Protein

As discussed above, the observation that the mitochondrial transmembranepotential (ΔΨm) was required for hSIRT3 import into mitochondriasuggested that hSIRT3 is likely to be imported across the innermitochondrial membrane. To further define the exact localization ofhSIRT3 in the mitochondria, we took advantage of established methodsaddressing the submitochondrial localization of proteins. First,mitochondria were isolated from HEK293T cells expressing hSIRT3-FLAG.Mitoplasts were prepared by incubation in hypotonic MOPS-buffer. Thistreatment leads to the rupture of the outer mitochondrial membrane andto the release of soluble proteins located in the intermembrane space.Mitoplasts and mitochondria were reisolated by centrifugation andanalyzed by western blotting (FIG. 5A). The ˜28 kDa form of hSIRT3 wasnot affected by the breakage of the outer mitochondrial membrane andsubsequent proteinase K digestion (FIG. 5A).

To exclude the possibility that hSIRT3-FLAG had formed a protease-stableaggregate, mitochondria from cells transfected with hSIRT3-FLAG werelysed in 0.5% Triton X-100 followed by proteinase K digestion. Underthese conditions, hSIRT3 was completely degraded. In this respect,hSIRT3 behaved in a manner similar to the matrix protein Hsp60 (FIG.5A). Confirmation of the rupture of the outer membrane by the hypotonictreatment was obtained by blotting against the intermembrane spaceprotein cytochrome c. In contrast to hSIRT3, cytochrome c was lost afterprotease treatment of mitoplasts (FIG. 5A). The results were consistentwith three different locations for hSIRT3: 1. mitochondrial matrix; 2.peripherally attached to the inner side of the inner mitochondrialmembrane; 3. integral inner mitochondrial membrane protein.

To differentiate between these possibilities, we performed alkalineextraction experiments of mitochondria with sodium carbonate at pH 11.5.This treatment releases soluble and peripheral membrane proteins to thesupernatant, while integral membrane proteins sediment with themembranes in the pellet (25). Following this treatment, the ˜28 KDa formof hSIRT3 was found in the supernatant, indicating that this form waseither a soluble matrix protein or was peripherally attached to theinner face of the inner membrane (FIG. 5A). Interestingly, the ˜44 KDaform of hSIRT3 was detected mostly in the pellet, suggesting that thisform of SIRT3 is associated with the inner mitochondrial membrane. Asexpected, the soluble matrix chaperonin mtHsp70 was detected in thesupernatant after alkaline extraction, whereas the inner-membraneprotein COXIV was associated with the membrane fraction (FIG. 5B). Theseexperiments indicate that the 28 KDa form or hSIRT3 is a soluble matrixprotein.

FIG. 5. A, Intramitochondrial localization of hSIRT3. Mitochondria wereisolated from hSIRT3-FLAG transfected HEK293T cells and treated withproteinase K (150 μg/ml) for 10 min at 0° C. to remove proteins bound tothe outer mitochondrial surface. Proteinase K treatment was stopped byincubation with 2 mM PMSF for 10 min at 0° C. Mitochondrial preparationswere divided and one half was diluted with hypotonic EM buffer to createmitoplasts. The other half was mock-treated with isotonic SEM buffer.After incubation for 20 min at 0° C., proteinase K (150 μg/ml) was addedfor 10 min at 0° C. Protease treatment was stopped as described aboveand mitochondria (M, left lane) and mitoplasts (MP, right lane) werereisolated and analyzed by western blotting. Opening of the outermitochondrial membrane was confirmed by detection of endogenousintermembrane space protein cytochrome c (cyt c). Integrity of the innermitochondrial membrane was determined using the matrix protein Hsp60 asa marker. hSIRT3-FLAG was detected using anti-FLAG M2 antibodies.

FIG. 5B, Alkaline extraction of mitochondria from hSIRT3-FLAGtransfected HEK293T cells. Mitochondria were isolated and treated withproteinase K (150 μg/ml) for 10 min at 0° C. PMSF (2 mM) was added tostop proteinase K digestion. Mitochondria were reisolated and washed inSEM buffer. The preparation was divided and one half was resuspended inSDS sample buffer (Total, left lane). The other half of the preparationwas resuspended in 100 mM sodium carbonate (Na₂CO₃), pH 11.5, andincubated for 30 min at 0° C. The extract was centrifuged at 100,000×gat 4° C. and the mitochondrial membranes (Pellet, middle lane) wereresuspended in SDS sample buffer. The supernatant containing the solubleand peripheral membrane proteins was TCA precipitated (Soluble, rightlane). Samples were analyzed by western blotting. hSIRT3 was detectedusing anti-FLAG antibodies. Alkaline extraction was controlled bydetection of the marker proteins COXIV and mtHsp70.

Proteolytic Processing of hSIRT3

As discussed above, the majority of hSIRT3 is present in mitochondria asa truncated ˜28 KDa protein. Since this form is reactive to theanti-FLAG antibody after transfection of a C-terminal FLAG fusionprotein, we concluded that hSIRT3 is proteolytically cleaved at itsN-terminus. The majority of mitochondrial proteins carrying N-terminaltargeting signals is processed by matrix processing peptidase (MPP)after import into the mitochondrial matrix (38). Incubation ofradiolabelled hSIRT3 with recombinant yeast MPP led to its cleavage to aproduct of ˜28 KDa, undistinguishable in size from the product detect invivo in mitochondria (FIG. 6A). Cleavage of a fusion protein betweensubunit 9 of F0/F1-ATPase and DHFR (Su9-DHFR) by MPP in vitro resultedin the appearance of digestion products similar to what has beenpreviously reported (28). Based on the size of the processed hSIRT3protein, we scanned the primary sequence of hSIRT3 for putative MPPrecognition motifs. MPP specifically processes many mitochondrialprecursor proteins but no consensus processing site has emerged.However, an arginine at −2 relative to the cleavage site and additionalaromatic or hydrophobic residues in position 1 relative to the cleavagesite appear necessary for cleavage (39-41).

Several hSIRT3 mutants targeting arginine residues at positions 99, 100,133, 135, 139 and 158 were constructed by site-directed mutagenesis andsynthesized in rabbit reticulocyte in the presence of [³⁵S]-methionine.A mutant carrying two glycines substituted for arginines at position 99and 100 showed abrogation of cleavage by MPP in vitro (FIG. 6B), whileother mutants were unaffected. These results indicate that residuesR99/100 are critical for the processing of hSIRT3 by MPP. Transfectionof this construct into mammalian cells led to a partial inhibition ofthe processing of hSIRT3 into the 28 KDa fragment and a new fragment ofhigher molecular weight was detected.

Catalytic Activation of a Latent hSIRT3 by MPP-Mediated ProteolyticProcessing

It was noted that the in vitro translated hSIRT3 protein wascatalytically inactive in our in vitro deacetylase assay. Similarly,hSIRT3 expressed in E. coli was not processed and was poorly activeenzymatically. The hypothesis that proteolytic processing of hSIRT3might lead to its catalytic activation was tested. Unlabeled hSIRT3 wassynthesized in vitro using rabbit reticulocyte lysate. Samples weresplit in half and subjected to cleavage by recombinant MPP in vitro.Reactions were diluted and hSIRT3 was immunoprecipitated and assayed fordeacetylase activity in the presence or absence of NAD. Remarkably, thehSIRT3 processed by MPP showed NAD-dependent deacetylase activity,whereas the full-length uncleaved hSIRT3 remained inactive (FIG. 6C).These results linked processing of hSIRT3 to the activation of itsNAD-dependent deacetylase activity. To control that no unspecificfactors or MPP itself had caused the observed NAD-dependent deacetylaseactivity, we used the catalytic inactive hSIRT3-H248Y mutant. When thismutant was assayed in the same way as hSIRT3, no NAD-dependentdeacetylase activity after incubation and cleavage with MPP (FIG. 6C,left and right panels). These results demonstrate that proteolyticprocessing of hSIRT3 by MPP leads to the activation of its latentenzymatic activity.

FIG. 6. Proteolytic processing of hSIRT3 by MPP. FIG. 6A, Cleavage ofradiolabeled hSIRT3-FLAG (left panel) or pSu9-DHFR (right panel) wasassayed in HDM buffer in the presence or absence of purified recombinantyeast MPP (1 μl) for 45 min at 27° C. in a total volume of 20 μl.Samples were analyzed by SDS-PAGE and autoradiography. p, precursorform; m, mature form of pSu9-DHFR. FIG. 6B, Schematic illustration ofthe mutant showing abrogated MPP cleavage (upper panel). Radiolabeledwild-type hSIRT3-FLAG or hSIRT3R99/100G-FLAG were analyzed for MPPprocessing. Assay conditions were as described (see A). Efficiency ofproteolytic processing by recombinant yeast MPP was quantitated usingphosphorimaging (left panel). Autoradiography of the same experiment(right panel). FIG. 6C, MPP processing activates NAD-dependentdeacetylase activity of hSIRT3. Unlabeled hSIRT3-FLAG orhSIRT3H248Y-FLAG synthesized in rabbit reticulocyte lysate was incubatedwith recombinant yeast MPP or an equal amount of water for 45 min at 27°C. Samples were diluted with TXIP-1 buffer. FLAG-tagged proteins wereimmunoprecipitated with anti-FLAG M2 antibodies covalently bound toagarose for 2 hrs at 4° C. Immunoprecipitates were washed and analyzedfor in vitro deacetylase activity in the presence or absence of NAD (1mM). FIG. 6D, Western blot analysis of immunoprecipitates used in thedeacetylase assay.

Example 2 Enzymatically Active Recombinant SIRT 3 Protein

hSIRT3 is an NAD dependent, class III HDAC. SIRT3 localizes to themitochondrial matrix via an amphipathic α-helix rich NH₂-terminal. Oncein the mitochondrial matrix, hSIRT3 is proteolytically cleaved bymitochondrial matrix processing peptidase (MPP) between residues Ser101and Ile102. Full length hSIRT3 is enzymatically inactive, but exhibitsHDAC activity in vitro after MPP cleavage. Based on these observations,it was predicted that a recombinant form of SIRT3 lacking the first 100amino acids to mimic the cleavage that occurs in the mitochondria wouldbe active as an HDAC.

Cloning Strategy

PCR primers were designed to amplify hSIRT3 from Ser101 to Lys399 usinga pcDNA3.1-SIRT3-Flag plasmid (pEV821) as a template. Primer sequence isas follows: forward—GTGAATTCATATCTTTTTCTGTGGGTGC (SEQ ID NO:07),reverse—GTGAATTCGCCCTTGAATCATC (SEQ ID NO:08). Both primers included anEcoR1site so the PCR amplicon could be digested with EcoR1 for subcloniginto other vectors. The following PCR parameters were used: 94° C.-5′,(94° C.-30″, 55° C.-30″, 72° C.-60″)×30 cycles, 72° C.-7′. The EcoR1digested amplicon was then subcloned into the expression vector pTrcHis.Frame form B of pTrcHis was used for subcloning to express an in frame,amino terminal 6× His tagged SIRT3 (101-399 aa) recombinant protein.

Expression and Purification

DH5α bacteria were transformed with the pTrcHis-SIRT3(101-399) plasmid(pEV1453). Transformed bacteria were induced with 1.0 mM IPTG at 37 Cfor 2 h. The resulting 6×His-tagged protein was purified under nativeconditions at 4° C. by Ni-NTA affinity chromotography (Qiagen).

First, bacteria were pelleted and cleared lysate was prepared undernative conditions. The pellet was resuspended (50 mM NaH₂PO₄, pH8.0; 300mM NaCl; 10 mM imidazole) and incubated on ice for 30 minutes in thepresence of 1 mg/ml lysozyme. This mixture was then sonicated on ice(four 10-15 second bursts at 40-60% power) and centrifuged at 4° C. at14,000 rpm for 25 minutes. Supernatant (cleared lysate) was bound toNi-NTA resin (batch method, Qiagen) on a rotary mixer at 4° C. for 60minutes. Batch mixture was loaded into poly prep column (BioRad) andflow-through collected. The resin bed was washed twice with 4 ml of washbuffer (50 mM NaH₂PO₄, pH8.0; 300 mM NaCl; 20 mM imidazole), and thetagged proteins eluted 4 times with 0.5 ml elution buffer (50 mMNaH₂PO₄, pH8.0; 300 mM NaCl; 250 mM imidazole). SDS-PAGE analysisrevealed the second elution fraction contained a majority of recombinantprotein along with contaminating proteins. A concentrating spin column(Vivaspin 6) was used to concentrate the recombinant protein and removeexcess imidazole.

Activity Assay

Recombinant SIRT3 (1.5 μg) was resuspended in 100 μL deacetylase buffer(50 mM Tris-HCl [pH 9.0], 4 mM MgCl₂, and 0.2 mM DTT) with differentconcentrations of NAD (Sigma) and 20,000 cpm of the acetylated peptidesubstrate (in vitro acetylated histone H4 peptide).

The results are shown in FIG. 8. SIRT3 showed a dose-dependent HDACactivity similar to the activity demonstrated for SIRT2. This experimentdemonstrate that recombinant SIRT3 can function as a deacetylase invitro and offers a new tool for the screening of SIRT3 inhibitors andthe study of its enzymatic activity.

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While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. An in vitro method of identifying an agent that modulates an enzymatic activity of a human mitochondrial NAD-dependent deacetylase, the method comprising: contacting a mitochondrial NAD-dependent deacetylase polypeptide with a test agent in an assay mixture that comprises NAD and an acetylated histone peptide; and determining the effect, if any, of the test agent on the enzymatic activity of mitochondrial NAD-dependent deacetylase.
 2. The method of claim 1, wherein the human mitochondrial NAD-dependent deacetylase polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:01.
 3. The method of claim 1, wherein the acetylated histone peptide comprises amino acids 1-22 of histone
 4. 4. The method of claim 1, wherein the acetylated histone peptide contains a ¹⁴C-labeled acetyl group, and determining the effect of the agent on the enzymatic activity of the deacetylase is performed by measuring release of the radioactive acetyl group.
 5. The method of claim 1, wherein determining the effect of the agent on the enzymatic activity of the deacetylase is performed by detecting binding of an antibody specific for acetylated histone.
 6. An in vitro method for identifying an agent that modulates a level of mitochondrial NAD-dependent deacetylase in a cell, the method comprising contacting a cell that produces mitochondrial NAD-dependent deacetylase with a test agent; and determining the effect, if any, of the test agent on the level of mitochondrial NAD-dependent deacetylase.
 7. The method of claim 6, wherein determining the effect of the agent on the level of the deacetylase is performed by determining a level of mitochondrial NAD-dependent deacetylase mRNA in the cell.
 8. The method of claim 6, wherein determining the effect of the agent on the level of the deacetylase is performed by determining a level of mitochondrial NAD-dependent deacetylase polypeptide in the cell.
 9. A biologically active agent identified by a screening method according to claim 1 or claim
 6. 10. A pharmaceutical composition comprising a biologically active agent that reduces a level or an activity of a mitochondrial NAD-dependent deacetylase protein; and a pharmaceutically acceptable excipient.
 11. A method of treating a disorder caused by mitochondrial malfunction or dysfunction in an individual, the method comprising administering to the individual an effective amount of an agent that modulates a level or activity of a mitochondrial NAD-dependent deacetylase protein.
 12. The method of claim 11, wherein the level or activity of the mitochondrial NAD-dependent deacetylase protein is increased.
 13. The method of claim 11, wherein the level or activity of the mitochondrial NAD-dependent deacetylase protein is decreased. 